5.1 materials materials source -...

Materials and methods

Dept of Pharmaceutics JSSCP, Mysore Page 70

5.1 Materials

Materials Source

Miconazole nitrate Bhavani Pharmaceuticals, Kanpur

5-Fluouracil Strides Arcolab, Bangalore

Chitosan Marine Chemicals, Cochin

Carbopol 71G Arihant Trading Co, Mumbai.

Carboxymethyl tamarind Creative polymer industries, Ananthpur

Polycarbophil/Noveon AA-1 Arihant Trading Co, Mumbai.

Sodium alginate Loba Chemie, Mumbai.

Microcrystalline cellulose Loba Chemie, Mumbai.

Talc Loba Chemie, Mumbai.

Sodium deoxycholate Sigma-Aldrich, Bangalore.

Potassium dihydrogen ortho

Phosphate

Loba Chemie, Mumbai.

Sodium hydroxide pellets Reachem Lab, India.

Methanol Loba Chemie, Mumbai.

Tween 80 Merck Specialities Pvt Ltd, Mumbai.

Sodium chloride Merck Specialities Pvt Ltd, Mumbai

Potassium hydroxide Spectrum reagent and chemical Pvt Ltd,

cochin.

Calcium hydroxide Loba chemie, Mumbai.

Bovine serum albumin Loba chemie, Mumbai.

Lactic acid Ranbaxy laboratories limited, Punjab.



Acetic acid Merck Specialities Pvt Ltd, Mumbai.

Glycerol Merck Specialities Pvt Ltd, Mumbai.

Urea Loba Chemie, Mumbai

Glucose Merck Specialities Pvt Ltd, Mumbai.

Hydrochloric acid Rankem, New Delhi



5.2 Instruments and Equipments

Equipments / Instruments

Source

Electronic balance Shimadzu Corporation, Japan.

FT – IR Spectrophotometer

Shimadzu, 8400S, Japan.

Dissolution apparatus

(8 basket)

Model No TDT-08L, Electrolab,

Mumbai, India

Orbital shaking incubator

Remi , Mumbai, India

UV-Visible Spectrophotometer

UV1800, Shimadzu, Japan

X-ray diffractometer

Miniflex II Desktop, Rigaku Corporation,

Japan

Hardness tester

Erweka .Germany

Micrometer screw gauge

Mitotoyo, Japan.

Friabilator

Electrolab, EF-2, Mumbai, India

Hot air oven Tempo instruments Pvt Ltd,Mumbai

Magnetic stirrer

Remi, Mumbai.



METHOD OF STUDY

Analytical method

Analytical method of miconazole nitrate (MN) in buccal pH 6.8 and simulated

vaginal pH 4.2

Analytical method of 5-fluorouracil (5-FU) in buccal pH 6.8, simulated vaginal

pH 4.2 and rectal pH 7.4

Preformulation studies

Solubility

Partition co-efficient

Drug excipients compatibility studies

INTERPOLYELECTROLYTE COMPLEX (IPEC)

Preparation and characterization

PREPARATION OF TABLET FORMULATION

Formulation of tablets using Chitosan- Carbopol 71G IPEC

Formulation of tablets using Chitosan-carboxymethyltamarind IPEC

Formulation of tablets using Chitosan- Polycarbophil IPEC

Formulation of tablets using chitosan- Sodium alginate IPEC

EVALUATION OF THE TABLET

Physicochemical properties

Swelling studies

In vitro drug release

In vitro mucoadhesive studies

In vivo studies

Ex vivo permeation studies

Stability studies



5.3 Preparation of buffer solutions

Phosphate buffer (pH 6.8)

50 ml of 0.2M potassium di hydrogen phosphate was taken in 200 ml volumetric flask, to

which 22.4 ml of 0.2 M sodium hydroxide solution was added and the volume was made

up to the mark with distilled water[154].

Simulated vaginal fluid

Simulated vaginal fluid (SVF) was prepared as reported: 3.51 g/l NaCl, 1.40 g/l KOH,

0.222 g/l Ca(OH)2, 0.018 g/l bovine serum albumin (BSA), 2 g/l lactic acid, 1 g/l

CH3COOH, 0.16 g/l glycerol, 0.4 g/l urea and 5 g/l glucose. The pH was correct at 4.2

with HCl 0.1N [155].

Phosphate buffer (pH 7.4)

Potassium dihydrogen phosphate of 0.2 M (50 ml) and 39.1 ml of 0.2 M NaOH were

taken in a 200 ml volumetric flask and made up to the volume with water[154].

0.2M potassium dihydrogen phosphate

27.218 gm of potassium dihydrogen phosphate was added to 1000 ml volumetric flask

containing distilled water and the volume was made up to the mark with distilled water.

0.2M sodium hydroxide

8 gm of NaOH was taken in a 1000 ml volumetric flask containing distilled water and

volume was made up to the mark with distilled water.



5.4. ANALYTICAL METHODS

5.1. Estimation of Miconazole nitrate (MN)

Determination of max of MN in phosphate buffer pH 6.8 (Buccal pH)

Preparation of stock solution

Accurately weighed 100 mg of MN was dissolved in small amount of methanol

and then 1 % of tween 80 was added. The volume was made up to 100 ml using the

phosphate buffer pH 6.8.

Scanning

From the stock solution, 100-600 mcg / ml solutions were prepared by pipetting

1-6 ml to a series of 10 ml volumetric flasks and the volume was made up to 10 ml with

phosphate buffer pH 6.8. The UV scan of these solutions was taken between 400-200

nm. The absorption maximum of MN was found to be 272 nm and this wavelength was

used for further studies. The spectrum is shown in Figure 2. The calibration curve data

are given in Table 1 and calibration curve is shown in the Figure 11.

Figure 10: UV spectra of MN in phosphate buffer pH 6.8


Dept of Pharmaceutics JSSCP, Mysore 6

Table 1: Calibration curve data of MN in phosphate buffer pH 6.8

Sl.

No.

Concentration

in μg/ml

Absorbance

± S.D Mean*

1 100 0.1378±0.0005

2 200 0.2724±0.0001

3 300 0.4002±0.0006

4 400 0.5245±0.0003

5 500 0.6460±0.0006

6 600 0.7892±0.0006

* Standard deviation n = 3

Figure 11: Calibration curve of MN in phosphate buffer pH 6.8



Determination of max of MN in simulated vaginal fluid (SVF) pH 4.2 (Vaginal pH)


Accurately weighed 100 mg of MN was dissolved in small amount of methanol

and then 1 % of tween 80 was added. The volume was made up to 100 ml using the SVF

pH 4.2.

Scanning


0.25-2 ml to a series of 10 ml volumetric flasks and the volume was made up to 10 ml

with phosphate buffer pH 6.8. The UV scanning of these solutions was taken between

400-200 nm. The absorption maximum of MN was found to be 271 nm and this

wavelength was used for further studies. The spectrum is shown in Figure 12. The

calibration curve data are given in Table 2 and calibration curve is shown in the Figure

13.

Figure 12: UV spectra of MN in SVF pH 4.2



Table 2: Calibration curve data of MN in SVF pH 4.2

Sl.

No.

Concentration

in μg/ml

Absorbance

± S.D Mean*

1 25 0.1093±0.0002

2 50 0.2273±0.0005

3 75 0.3225±0.0004

4 100 0.4467±0.0002

5 125 0.5499±0.0003

6 150 0.6658±0.0006

7 175 0.7842±0.0006

8 200 0.9153±0.0002


Figure 13: Calibration curve of MN in SVF pH 4.2



5.4.2 Estimation of 5-fluorouracil (5-FU)

Determination of max of 5-Fluorouracil (5-FU) in phosphate buffer pH 6.8.


Accurately weighed 100 mg of 5-FU was dissolved in small amount of distilled water.

The volume was made up to 100 ml using the phosphate buffer pH 6.8.

Scanning


0.2-1 ml to a series of 10 ml volumetric flasks and the volume was made up to 10 ml with

phosphate buffer pH 6.8. The UV scanning of these solutions was taken between

400-200 nm. The absorption maximum of 5-FU was found to be 266 nm and this


calibration curve data are given in Table 3 and calibration curve is shown in the Figure

15.

Figure 14: UV spectra of 5-FU in phosphate buffer pH 6.8



Table 3: Calibration curve data of 5- FU in phosphate buffer pH 6.8

Sl. No.

Concentration

(mcg/ml)

Absorbance

± S.D Mean*

1 2 0.1777±0.0001

2 4 0.3221±0.0002

3 6 0.5021±0.0002

4 8 06715±0.0002

5 10 0.8214±0.0001


Figure 15: Calibration curve of 5-FU in phosphate buffer pH 6.8



Determination of max of 5-FU in simulated vaginal fluid (SVF) pH 4.2.


Accurately weighed 100 mg of 5-FU was dissolved in small amount of distilled water.

The volume was made up to 100 ml using the SVF pH 4.2.

Scanning


0.5-2.5 ml to a series of 10 ml volumetric flasks and the volume was made up to 10 ml

with simulated vaginal fluid pH 4.2. The UV scanning of these solutions was taken

between 400-200 nm. The absorption maximum of 5-FU was found to be 265 nm and

this wavelength was used for further studies. The spectrum is shown in Figure 16. The

calibration curve data is given in Table 4 and calibration curve is shown in the Figure 17.

Figure 16: UV spectra of 5-FU in SVF pH 4.2



Table 4: Calibration curve data of 5-FU in SVF pH 4.2

Sl. No.

Concentration

(in mcg/ml)

Absorbance

± S.D Mean*

1 5 0.1831±0.0001

2 10 0.3471±0.0001

3 15 0.5151±0.0002

4 20 0.6723±0.0001

5 25 0.8330±0.0002


Figure 17: Calibration curve of 5-FU in SVF pH 4.2



Determination of max of 5-FU in phosphate buffer pH 7.4 (Rectal pH)


Accurately weighed 100 mg of 5-FU was dissolved in a small amount of distilled water.

The volume was made up to 100 ml using the phosphate buffer pH 7.4.

Scanning


1-4 ml to a series of 10 ml volumetric flasks and the volume was made up to 10 ml with

phosphate buffer pH 7.4. The UV scanning of these solutions was taken between

400-200 nm. The absorption maximum of 5-FU was found to be 267 nm and this


calibration curve data is given in Table 5 and calibration curve is shown in the Figure 19.

Figure 18: UV spectra of 5-FU in phosphate buffer pH 7.4



Table 5: Calibration curve data of 5-FU in phosphate buffer pH 7.4

Sl. No.

Concentration

(in mcg/ml)

Absorbance

Mean± S.D*

1 3 0.1591±0.0001

2 6 0.3220±0.0001

3 9 0.4531±0.0003

4 12 0.6121±0.0002

5 15 0.7751±0.0002


Figure 19: Calibration curve of 5-FU in phosphate buffer pH 7.4



5.5 PREFORMULATION STUDIES

Solubility

Excess amount of MN was shaken with 2 ml of buffer solution (phosphate buffer pH 6.8

and SVF pH 4.2 separately) at room temperature, until equilibrium was reached.

Similarly for 5-FU was shaken in phosphate buffer pH 6.8, SVF 4.2 and pH 7.4

separately. The solution was then filtered and the concentration of drug in solution was

determined by U.V spectroscopic method using shimadzu 1800 U.V visible

spectrophotometer [156].

Partition coefficient

Mutually saturated 1- octanol and phosphate buffer solution (pH 6.8) at 37 °C was used

for the study. An aliquot (10 ml) of 1-octanol saturated phosphate buffer solution

containing suitable concentration of the 5-FU (100 mcg/m1) was mixed with an equal

volume of phosphate buffer (pH 6.8) saturated 1-octanol. Two phases were then allowed

to equilibrate at 37 °C for 24 hrs on a magnetic stirrer. Similarly in SVF fluid pH 4.2 and

pH 7.4 was carried out [157].

The concentration of the drug in the aqueous phase was determined by U.V

Spectroscopic method. The apparent partition coefficient (kp) was calculated as the ratio

of drug concentration in each phase by the following equation.

Caq-Ceq

Kp =

Ce



Where, Caq is initial concentration of drug in aqueous phase and Ceq is the concentration

of drug at equilibrium in aqueous phase. Phosphate buffer pH 6.8, dissolved in ocantol

phase, is not taken into account in this measurement.

Drug–Excipient compatibility

In order to ascertain whether or not any interaction occurred between the polymers and

drug substances, the characterization of drug, polymer and physical mixture of drug:

polymer have been done using differential scanning calorimetry (DSC) and fourier

transform infrared spectroscopy (FT-IR).

Differential scanning calorimetry: All the dynamic DSC studies were carried out on

DSC 50, Shimadzu Scientific Instruments, Japan. Calorimetric measurements were made

with empty cell (high purity alpha alumina discs) as the reference. The instrument was

calibrated using high purity indium metal as standard. The dynamic scans were taken in

nitrogen atmosphere at a heating rate of 10 °C min. The runs were made in triplicate.

Fourier Transform Infrared (FT-IR) Spectroscopy: The test sample was dispersed in

KBr powder and analyzed. FT-IR spectra were obtained by diffuse reflectance on a

FT-IR spectrophotometer type FT-IR 8400S shimadzu, Japan. Compatibility between the

drugs and the polymers were compared by FT-IR spectra. The positions of FT-IR bands

of important functional groups of drugs were identified and were cross checked with FT-

IR spectra of drug with excipients in 1:1ratio.



Formulation design overview

Figure 20: Schematic representation of formulation design

PM: Physical mixture, IPEC: Interpolyelectrolyte complex, CMT: Carboxymethyl

tamarind, SDC: Sodium deoxycholate

Part I (14 formulation)

Polymers used Formulation code

Chitosan MA1,MA2

Carbopol 71G MB1,MB2

Chitosan-carbopol

71G PM

MC1, MC2

Chitosan-carbopol

IPEC

MD1,MD2, MD3

MD4

IPEC, chitosan and

carbopol

ME1, ME2,ME3

ME4

Part II (12 formulations)


CMT MF1,MF2

Chitosan- CMT

PM

MG1,MG2

Chitosan-CMT IPEC MH1,MH2,MH3,

MH4

IPEC, chitosan and

CMT

MI1,MI2,MI3,

MI4

Part III (12 formulations)


Polycarbophil FA1,FA2

Chitosan-

Polycarbophil PM

FB1,FB2

Chitosan-

Polycarbophil IPEC

FC1,FC2,FC3,

FC4

IPEC, chitosan and

polycarbophil

FD1,FD2

IPEC, chitosan,

Polycarbophil,

SDC

FE1,FE2

Part IV (12 formulations)


Sodium alginate FF1,FF2

Chitosan-sodium

alginate PM

FG1,FG2

Chitosan-sodium

alginate IPEC

FH1,FH2,FH3,

FH4

IPEC, chitosan and

sodium alginate

FI1,FI2

IPEC, chitosan,

Sodium alginate,

SDC

FJ1,FJ2

Miconazole nitrate

(MN)

5-fluorouracil

(5-FU)

Design for oral

and vaginal

candidiasis

Design for oral,

cervical and

colorectal

cancer



5.6. FORMULATION DESIGN OF MICONAZOLE NITRATE TABLETS

Chitosan-carbopol 71G IPEC

5.6.1 Preparation of chitosan- carbopol 71G interpolyelectrolyte complex (IPEC)

A Carbopol 71G aqueous solution (1 mg/ml) and chitosan aqueous acetic acid solution

(5 mg/ml) were mixed. The resulting precipitate (carbopol/chitosan IPEC) was washed

with distilled water and filtered under vacuum pump. The filtrate was dried in hot air

oven and the dried complex was ground with a grinder. The powder was passed through a

200µm sieve and used for further study.

5.6.2 Turbidity measurement of chitosan- carbopol IPEC ratios

The carbopol 71G/chitosan ratio in the complex was examined by monitoring the

transmittance of the solution at a wavelength of 600 nm using a spectrophotometer

(UV-1800, Shimadzu, Japan). An aqueous carbopol 71G solution (0.5, 1, 1.5, 2, 2.5, 3,

3.5, and 4 mM) and a chitosan aqueous acetic acid solution (0.5, 1, and 2 mM) were used.

The concentration was calculated by dividing the weight of chitosan and carbopol by the

formula weight of each monomer unit. Each mixture was shaken vigorously. The

mixtures were kept aside for 10 min before measuring the transmittance as a function of

the various mixing ratios (chitosan/ carbopol 71G) [158].

5.6.3 Preparation of miconazole nitrate tablets

Mucoadhesive tablets were fabricated by direct compression method as shown in Table 6.

The accurate quantity of miconazole nitrate and excipients was weighed. They were

passed through sieve # 100 and thoroughly mixed using mortar and pestle. The blend was



lubricated and then compressed into compacts by direct compression method using 8-mm

flat-faced punches in KBr press (Technosearch, Mumbai, India) at 1 ton pressure with a

dwell time of 1 s.

Table 6: Formulation chart for MN matrix tablets

Formulation

code

Chitosan

(mg)

Carbopol

71G(mg)

Chitosan-

carbopol 71G

physical

mixture (1:1)

(mg)

IPEC

(mg)

MCC

(mg)

Talc

(mg)

MA1 50 --- --- ---- 45 5

MA2 100 --- --- ---- ---- ---

MB1 --- 50 --- --- 45 5

MB2 --- 100 --- --- --- ---

MC1 --- --- 50 --- 45 5

MC2 --- --- 100 -- --- ---

MD1 --- --- --- 60 35 5

MD2 --- --- --- 70 25 5

MD3 --- --- --- 80 15 5

MD4 --- --- --- 90 5 5

ME1 20 --- --- 70 5 5

ME2 25 --- --- 65 5 5

ME3 10 10 --- 70 5 5

ME4 15 15 --- 60 5 5

Miconazole nitrate is 50mg

Total weight of tablet is 150 mg



Chitosan- carboxymethyl tamarind IPEC

5.6.4 Preparation of chitosan/carboxymethyl tamarind interpolyelectrolyte complex

(IPEC)

2 % w/v Chitosan aqueous acetic acid solution and 2 % w/v of Carboxymethyl tamarind

solution (CMT) were mixed under homogenization under 1000 rpm. The resulting

precipitate (Chitosan/CMT IPEC) was washed with distilled water and filtered under

vacuum pump. The filtrate was dried in hot air oven and the dried complex was ground

with a grinder. The powder was passed through a 200 µm sieve and used for further study

[159].

5.6.5 Viscosity study of chitosan-CMT IPEC ratios

Chitosan solution was prepared in 2 % v/v acetic acid. CMT solutions were separately

prepared by hydrating them in distilled water. Both the solutions were at 25 °C to obtain

different ratios of CH: CMT. The samples were incubated at 37 °C for 24 h. The samples

were then centrifuged at 15000 rpm. The viscosity of the supernatant solution was

determined using Brookfield RVDV II Pro Viscometer, UK (Spindle 21).

5.6.6 Preparation of miconazole nitrate tablets using chitosan-CMT IPEC


The accurate quantity of miconazole nitrate and excipients was weighed. They were

passed through sieve # 100 and thoroughly mixed using mortar and pestle. The blend was



dwell time of 1 s.



Table 7: Formulation chart for MN matrix tablet

Ingredients MF1 MF2 MG1 MG2 MH1 MH2 MH3 MH4 MG1 MG2 MG3 MG4

MN (mg) 50 50 50 50 50 50 50 50 50 50 50 50

CMT(mg) 50 100 --- --- ---- --- ---- --- 20 20 30 20

Chitosan–

CMT

physical

mixture

(1:1) (mg)

---- ----- 50 100 ---- --- --- --- --- --- ---

IPEC (mg) ------ ---- --- --- 60 70 80 90 80 60 60 70

Chitosan

(mg) --- --- --- --- --- --- --- --- -- 20 10 10

MCC (mg) 45 ---- 45 --- 35 25 15 5 -- --- -- --

Talc (mg) 5 ---- 5 --- 5 5 5 5 -- --- -- ---




5.7. Formulation Design for 5-FU tablets

Chitosan-polycarbophil IPEC

5.7.1 Preparation of chitosan-polycarbophil interpolyelectrolyte complex (IPEC)

A 3 % w/v Chitosan solution and a 3 % w/v of polycarbophil solution were

prepared separately in solutions of 2 % v/v acetic acid. The chitosan solution was added

slowly to the polycarbophil solution under homogenization at 1000 rpm over a period of

20 min. the mixture was then stirred for a period of 1 h at a speed of 900 rpm with digital

mechanical stirrer. The resulting precipitate (Chitosan- polycarbophil IPEC) was washed

several times with 2 % v/v acetic acid solution to remove any noncomplexed polymeric

material and filtered under vacuum pump. The product was dried in hot air oven and the

dried complex was powdered with a grinder. The powder was passed through a 200 µm

sieve and used for further study.

5.7.2 Transmittance measurement of chitosan-polycarbophil IPEC ratios

Transmittance measurements were carried out with a UV spectrophotometer (Shimadzu

UV visible 1800, Shimadzu Scientific Instruments, Japan) at the wavelength λ= 420 nm

for supernatant liquids of various concentration of chitosan-polycarbophil IPEC

[160,161].Solutions of 3 % (w/v) of chitosan and various concentration of polycarbophil

solution ranging from 0.5 to 5 % (w/v) in 2 % acetic acid solution (v/v) were prepared

separately. Then the chitosan solution was slowly added to polycarbophil solution and

kept aside. The resultant solution was filtered and the supernatant liquid of the solution

was subjected for transmittance.



5.7.3 Preparation of 5-fluorouracil tablets using chitosan-polycarbophil IPEC


The accurate quantity of 5-fluorouracil and excipients was weighed. They were passed

through sieve # 100 and thoroughly mixed using mortar and pestle. The blend was



dwell time of 1 s.

Table 8: Formulation chart for 5-FU matrix tablet

Ingredients FA1 FA2 FB1 FB2 FC1 FC2 FC3 FC4 FD1 FD2 FE1 FE2

5-Fluorouracil

(mg)

20 20 20 20 20 20 20 20 20 20 20 20

Polycarbophil

(mg)

60 80 --- ----- ---- ----- ----- ----- 10 20 20 20

Chitosan-

polycarbophil

physical

mixture(1:1)

(mg)

---- ----- 80 100 --- ---- ----- ----- ----- ---- --- ---

IPEC (mg) -----

-

----- ---- --- 40 60 80 100 80 80 80 80

Chitosan (mg) ---- ---- ---- ----- ---- ---- ---- --- 10 20 20 20

SDC (mg) ---- ---- ----- ----- --- ---- ---- ---- ---- ---- 3 4.5

MCC (mg) 65 45 45 25 85 65 45 25 25 5 2 0.5

Talc (mg) 5 5 5 5 5 5 5 5 5 5 5 5




Chitosan- sodium alginate IPEC

5.7.4 Preparation of chitosan/sodium alginate interpolyelectrolyte complex (IPEC)

The chitosan–alginate polyelectrolyte complex was prepared from chitosan solution at

4.0 % w/v in 2 % w/w acetic acid solution and sodium alginate solution at 4.0 % w/v in

water. Both solutions were heated separately at 70–80 °C. Both solutions were mixed

with agitation until the mixture reached room temperature. Then it was kept aside for 2 h.

The interpolyelectrolyte complex (IPEC) was thoroughly washed with distilled water and

then separated from water by centrifugation for 30 min at 10000 rpm. Thereafter IPEC

was dried in hot air oven and the dried complex was powdered with a grinder. The

powder was passed through a 200 µm sieve and used for further study [162].

5.7.5 Viscosity study of chitosan-alginate IPEC ratios

Chitosan solution was prepared in 2 % v/v acetic acid and sodium alginate solution was

prepared in water. Both the solutions were mixed together to obtain different ratios of

IPEC. The samples were incubated at 37 °C for 24 h. The samples were then centrifuged

at 15000 rpm. The viscosity of the supernatant solution was determined using Brookfield

RVDV II Pro Viscometer, UK (Spindle 21).

5.7.6 Preparation of 5-fluorouracil tablets using chitosan-sodium alginate IPEC


The accurate quantity of 5-fluorouracil and excipients was weighed. They were passed

through sieve # 100 and thoroughly mixed using mortar and pestle. The blend was





dwell time of 1 s.

Table 9: Formulation chart for 5-FU matrix tablet

Ingredients FF1 FF2 FG1 FG2 FH1 FH2 FH3 FH4 FI1 FI2 FJ1 FJ2

5-Fluorouracil

(mg)

20 20 20 20 20 20 20 20 20 20 20 20

Sodium alginate

(mg)

40 80 --- ----- ---- ----- ----- ----- 10 20 20 20

Chitosan-

sodium alginate

physical

mixture(1:1)

(mg)

---- ----- 80 100 --- ---- ----- ----- ----- ---- --- ---

IPEC (mg) ------ ----- ---- --- 40 60 80 100 80 80 80 80

Chitosan (mg) ---- ---- ---- ----- ---- ---- ---- --- 10 20 20 20

SDC (mg) ---- ---- ----- ----- --- ---- ---- ---- ---- ---- 3 4.5

MCC (mg) 85 45 45 25 85 65 45 25 25 5 2 0.5

Talc (mg) 5 5 5 5 5 5 5 5 5 5 5 5




5.8 Evaluation

5.8.1 Characterization of IPEC

Fourier transform infrared (FT-IR) spectroscopy study

The infrared absorption spectra of polymers alone and their IPEC were analyzed using a

FT-IR spectrophotometer (shimadzu 8400S). The pellets were prepared by pressing the

sample with potassium bromide in the ratio of 1:100.

Differential scanning calorimetry (DSC)

Thermal analysis of only the polymers and there IPEC was carried out using a differential

scanning calorimeter (DSC 50, Shimadzu Scientific Instruments, Japan). The samples

were placed in an aluminum-sealed pan and preheated to 200 °C. The sample was cooled

to room temperature and then reheated from 40 to 400 °C at a scanning rate of 10 °C/min.

Powder X-ray Diffraction

Powder X-ray diffraction patterns on polymers alone and their IPEC were obtained by

using an X-ray Diffractometer (Miniflex II Desktop X-ray Diffractometer, Rigaku

Corporation, Tokyo, Japan). The samples were scanned from 6° to 40° (2θ) with an

increment of 0.02° and measurement time of 10 s/increment.

5.8.2 Evaluation of tablets

5.8.2.1 Physicochemical properties

Determination of drug content

The prepared formulations were analyzed for miconazole nitrate content, by taking

150 mg of tablet powder in 100 ml volumetric flask, to which methanol was added and

shaken well. Further, the volume was made up to the mark with methanol. The drug



content was determined by measuring the absorbance at 272 nm using UV-

spectrophotometer. Similarly 5-fluorouracil was determined in distilled water at 265 nm

using UV spectrophotometer.

Determination of weight variation

Twenty tablets were randomly selected from each batch and individually weighed. The

average weight and standard deviation of 20 tablets was calculated. The batch passes the

test for weight variation test, if not more than two of the individual tablet weight deviate

from the average weight by more than the percentage shown in table 10.

Table 10: Maximum % deviation allowed as per IP

Average weight Maximum % deviation

allowed

130 or less 10

130-324 7.5

More than 324 5

Determination of thickness

Twenty tablets were randomly selected from each batch and their thickness was measured

by using vernier calipers. It is expressed in millimeter.

Determination of hardness

The hardness of the tablet was determined using Inweka hardness tester. For each batch

three tablets were tested. It is expressed in Newton.

Determination of friability

Twenty tablets were weighed and placed in the friabilator. The apparatus was rotated at

25 rpm for 4 minutes. After revolutions the tablets were dedusted and weighed again. The

percentage friability was measured using the formula.



100

Initial

Finalinitial

W

WWF

5.8.2.2 Swelling studies

The swelling index of the prepared matrix tablets was determined by weighing five

tablets and recording their weights before placing them separately in weighed beakers.

The total weight was recorded (W1). Ten milliliters of phosphate buffer pH 6.8 (similarly

with simulated vaginal fluid of pH 4.2 and phosphate buffer pH 7.4) was added to each

beaker and then placed in an incubator at 37±0.5 °C. At time intervals of 2, 4, 6 and 8 h

excess water was carefully removed, and the swollen tablets were weighed (W2). The

experiment was repeated three times, and the difference of W1 and W2 was reported. The

percentage swelling index was determined using the formula.

Swelling index= W2-W1/W1*100

5.8.2.3 In vitro dissolution studies

The drug release rate from buccal tablets was studied using the orbital shaking incubator

using (Remi CIS 24, India) 30 mL of phosphate buffer pH 6.8. The temperature was

maintained at 37±0.5 °C and 50 rpm (rotation per min). For every one hour of time

interval, 3 mL sample was withdrawn and filtered through a Millipore filter of 0.45 µm

pore size and assayed spectrophotometrically at 272 nm for miconazole and 266 nm for

5-fluorouracil. Immediately after each sample withdrawal, a similar volume of phosphate

buffer pH 6.8 was added to the dissolution medium [163].

The drug release rates from vaginal tablets were studied in 500 ml of simulated vaginal

fluid pH 4.2 in type II dissolution apparatus. The temperature was maintained at

37±0.5 °C and 50 rpm. 10 mL sample was withdrawn at hourly interval, filtered through



a Millipore filter of 0.45 µm pore size and assayed spectrophotometrically at 271 nm for

miconazole and 265 nm for 5-fluourouracil. Immediately after each sample withdrawal,

a similar volume of simulated vaginal fluid pH 4.2 was added to the dissolution medium

[164].

In vitro drug release for rectal tablets was performed using the dissolution apparatus I;

500 mL phosphate buffer pH 7.4 maintained at 37± 0.5 °C was used as a dissolution

medium. Basket was rotated at 50 rpm. 10 mL aliquots were taken at periodic time

intervals and replaced by equal volume of phosphate buffer pH 7.4. The solution was

suitably diluted and the absorbance was taken at 267 nm for 5-fluorouracil using UV

visible spectrophotometer [147].

5.8.2.4 In vitro mucoadhesive studies

Mucoadhesive strength of the tablets was measured using modified physical balance. In

vitro bioadhesion studies were carried out using sheep buccal mucosa and modified two-

armed balance. The phosphate buffer pH 6.8 was used as the moistening fluid. A glass

stopper was suspended by a fixed length of thread on one side of the balance and was

counter balanced with the weights on the other side. Fresh sheep buccal mucosa was

collected from the slaughter house. It was scrapped off from the connective tissues and a

thin layer of buccal mucosa was separated and used for the bioadhesion study. A circular

piece of sheep buccal mucosa was cut and fixed to the tissue holder and immersed in

phosphate buffer pH 6.8 and the temperature was maintained at 37± 1 °C. Then the tablet

was fixed to a glass stopper with the help of cyanoacrylate adhesive and placed on the

buccal mucosa by using a preload of 50 gm and kept aside for 1 min to facilitate adhesion



bonding. After preloading time, the preload was removed and the weights were added on

the other side of the balance until tablet detaches from the sheep buccal mucosa. The

weight required to detach tablet from buccal mucosa was noted.

Figure 21: Modified physical balance for mucoadhesive studies

5.8.2.5 Ex vivo permeation study

Permeation study was carried out for the optimized 5-fluorouracil tablets using Franz

diffusion cell. The tablet was placed in the donor compartment on the sheep mucosa. The

mucosal layer is on donor compartment. The receptor compartment was filled with

phosphate buffer pH 6.8. The temperature was maintained at 37± 0.5 °C and 50 rpm. The

amount of 5-fluorouracil permeated through sheep mucosa was determined by

withdrawing 3 ml of aliquots from the receptor compartment using a syringe and

immediately replacing the same volume of solution.

5.8.2.6 Mathematical model fitting

The release data was fitted into various mathematical models using PCP Disso-

V2.08 software. The parameters like ‘n’ the time exponent, ‘k’ the release rate constant



and ’R’ the regression co-efficient were determined to know the release mechanisms. The

various models studied were:

First order

Zero order

Matrix model

Hixon-crowell model

Higuchi model

Peppas model fitting

The data obtained from in vitro release studies was put into Peppas model. The various

parameters viz., the intercept A, the release constant K and regression coefficient R2

were

calculated.

Koresmeyer-Peppas equation: Mt/M∞ = 1- A (exp -Kt)

Log (1 - Mt/M∞) = log A – kt/2.303

Where, Mt – Amount of drugs released at time t

M∞ – Total amount of drug released after an infinite time

K – Diffusion constant

A – The Intercept

5.9 In vivo X-ray studies

The animal experiment project was cleared and approved by Institutional animal ethical

committee, J.S.S. College of Pharmacy, Mysore (Code: 106/2011)

The study was performed on a healthy female rabbit, weighing between 1 and 1.5 kg. The

optimized formulation was selected in order to study in vivo performance of the

preparation. Optimized formulation was modified by adding 50 mg of x-ray grade barium

sulfate for miconazole tablets and 20 mg of X-ray grade barium sulfate for 5-fluorouracil

tablets. The prepared tablet was placed in the buccal mucosa of a healthy rabbit. During



the study, the rabbit was not allowed to eat or drink. The rabbit was exposed to X-ray

examinations and photographs were taken at 1st and 8

th h after administration of the

tablet. Similar procedure was followed for vaginal drug delivery for miconazole and

5-fluorouracil tablets. The rabbit was exposed to X-ray examinations and photographs

were taken at 1st and 8

th h after administration of the tablet. Similar procedure was

followed for rectal drug delivery for 5-fluorouracil tablets. The rabbit was exposed to

X-ray examinations and photographs were taken at 1st and 8

th h after administration of the

tablet.

5.10 Stability Studies

Stability is defined as the ability of particular drug or dosage form in a specific container

to remain within its physical, chemical, therapeutic and toxicological specification. Drug

decomposition or degradation occurs during stability, because of chemical alteration of

the active ingredients or due to product instability, lowering the concentration of the drug

in the dosage form. The stability of pharmaceutical preparation should be evaluated by

accelerated stability studies. The objective of accelerated stability studies is to predict the

shelf life of a product by accelerating the rate of decomposition, preferably by increasing

the temperature. The optimized formulation of buccal miconazole nitrate tablets was

selected for the stability studies. The accelerated stability studies was carried out

according to ICH guidelines by storing the samples at 25±2 ºC and 60±5 %RH,

30±2 ºC and 65±5 % RH and 40±2 ºC and 75±5 % RH for 6 months. Samples were

withdrawn on 0 day, 3 months, and 6 months and were analyzed for physical stability and

drug content.

5.1 materials materials source -...

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